Toxic effect of arsenic on ten rice varieties

Keywords:
Arsenic stress, Morphological parameters, SDS-PAGE, Molecular variation,
Oryza sativa L.

Abbreviations:
SDS PAGE- Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis.
ABSTRACT:

Rice is one of the most important cereal crops of developing countries and
the staple food of about 65% of the worlds population. The rice crops have been
greatly disturbed by the heavy metals. The present study deals with the toxic effect of
sodium arsenate on morphological and molecular variation through SDS-PAGE in 10
rice (Oryza sativa L.) varieties. Ten varieties of rice were grown under different
concentration (25 ppm, 50 ppm and 100 ppm) of sodium arsenate against control.
Morphological parameters like shoot length, root length, leaf area and biomass
showed marked differences among ten rice varieties. The proteins were separated
through SDS-PAGE gel electrophoresis and calculated their molecular weight. The
morphological and molecular variations induced in rice varieties by arsenic stress
provide a new insight leading to a better understanding of the heavy metal response
in plants.
011-016 | JRA | 2011 | Vol 1 | No 1

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www.jagri.info
Journal of Research in
Agriculture
An International Scientific
Research Journal
Authors:
John De Britto R, Mary
Sujin R, Steena Roshan
Sebastian

and Dharmar K.




Institution:
Plant Molecular Biology
Research unit, St. Xavier's
College (Autonomous),
Palayamkottai, 627 002,
Tamilnadu, India.






Corresponding author:
John De Britto A


Email:
bjohnde@yahoo.co.in






Web Address:
http://www.jagri.info
documents/AG0003.pdf.


Dates:
Received: 12 Oct 2011 Accepted: 19 Oct 2011 Published: 25 Oct 2011
Article Citation:
John De Britto R, Mary Sujin R, Steena Roshan Sebastian

and Dharmar K.
Toxic effect of arsenic on ten rice varieties.
Journal of Research in Agriculture (2011) 1: 011-016
Original Research
Journal of Research in Agriculture
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An International Scientific Research Journal


INTRODUCTION
Heavy metals constitute a heterogeneous
group of elements widely varied in their chemical
properties and biological functions (Holleman and
Wiberg, 1985). Heavy metals are kept under
environmental pollutant category due to their toxic
effects in plants, human and food. Some of the
heavy metals are cumulative poison especially
Arsenic (As), Cadmium (Cd), Lead (Pb) and
Mercury (Hg) (Mildvan, 1970). Metal toxicity to
plants varies with plants species, specific metals,
concentration, chemical form, soil composition and
pH (Mukesh et al., 2008). Off many, arsenic is one
among that drastically affects the crops.
The increased level of arsenic has been
mainly resulted from mining, industrial, agricultural
and geochemical processes (Flora et al., 2007).
Food crops such as rice and vegetables grown on
arsenic contaminated soil can accumulate high
levels of arsenic in roots, shoots and seeds (Zhu et
al., 2008). Thus arsenic uptake by crop plants plays
an important role in the transfer of this toxic
element into the food chain. Additionally, inorganic
arsenic species are phytotoxic and the elevated
concentration of arsenic in the soil causes a
significant reduction in crop yield (Meharg, 2004).
This stress may induce genetic changes in the crop
plants.
Rice is one the world's most important food
crops and grown mostly in tropical and subtropical
countries (Singh, 1993). The demand for rice is
increasing tremendously as the population in rice
growing areas is increasing at an alarming rate.
However, increasing rice production is becoming
more difficult because of biotic and abiotic stresses
(Hert, 1991).
In the present study, analysis of morphology
and protein variability of the ten rice varieties
induced with arsenic stress through SDS-PAGE has
been carried out.

MATERIALS AND METHODS
Materials
The seeds of rice varieties were procured
from seed testing centre, Palayamkottai. Ten
popular, medium duration varieties namely IR 32,
IR 36, IR 45, IR 46, IR 50, ADT 32, ADT 39, CO
45, Ambai 16 and Singapore were selected for the
present study.
Methodology
A pot culture experiment was conducted in
Plant Molecular Biology Research Unit, St.
Xaviers College, Tirunelveli. The seeds were
surface sterilized and soaked overnight in double
distilled water (ddw) and then kept in dark for 24 hr
for germination. Then these were sown in pots
(paper cup), which were watered adequately. After
10 days the samples were exposed to 25 ppm, 50
ppm and 100 ppm of arsenic solution (Na2HAsO4),
and a control was maintained.
Morphometric analysis
After 15, 20, 25 & 30
th
day, the shoot length,
leaf area were measured using a metric scale. On
the 30
th
day, the plants were harvested and
separated in to root and shoot. Length of root and
their fresh and dry weights were measured.
Protein profiling
The collected shoots were taken for protein
isolation. The separation of protein was carried out
at -50C at 100V thereafter for 3-5 hours. SDS-
PAGE electrophoresis preparation was followed by
(Laemmli, 1970).
2.4. Data analysis:
The obtained data was tabulated, the
molecular weight of protein was determined and
their banding patterns were analyzed.

RESULTS AND DISCUSSION
The effect of sodium arsenate metal on
morphological and molecular parameters in ten rice
varieties were carried out.
The plants were treated with different
concentrations of sodium arsenate (25, 50 and 100
ppm), which caused a reduced growth in shoot
length, leaf area, root length, fresh and dry weights
(Table 1). Regarding shoot and root length there
were significant difference in the height of the plant
treated with different sodium arsenate concentration
when compared to the control. The fresh and dry
weight of stressed plants reduced when compared to
control in response to arsenic stress.
In most of the varieties, the shoot length at
highest concentration remained the same for 20
th
,
25
th
and 30
th
days; whereas, the low concentrations
showed normal growth with an increase in shoot
length. Decrease in shoot length may be due to the
inhibition of cell division and cell enlargement. The
IR45 variety with shoot length 12.6 cm was more
tolerant to the arsenic stress, whereas ADT39
variety with shoot length 9 cm was less tolerant
among the ten rice varieties chosen.
Muhammad et al. (2008) reasoned that the
reduced shoot and root length in metal treated
seedlings could be due to the reduction in the
meristematic cells present in this region and some
enzymes contained in the cotyledon and endosperm
012 Journal of Research in Agriculture (2011) 1: 011-016
Britto et al.,2011
cells become active and begin to digest the stored
food, which is converted into soluble form and
transported to the radicle and plumule tips. Hence,
when activities of hydrolytic enzyme are affected,
the food does not reach the radicle and plumule
thereby affecting the seedling length.
Leaves of metal treated plants showed
damage symptoms such as chlorosis, necrosis, leaf
burn and senescence as well as leaf area reduction.
High concentration (100 ppm) of metal treatment
strongly reduced the leaf area of the IR46 variety at
30
th
day. The root length measured at 30
th
day
showed decrease in growth with respect to
increased metal concentration. Prasad (1995)
reasoned that the increased phytotoxicity with
increase in the concentration of heavy metals was
due to the accumulation of heavy metals in the root
which reduced the mitotic division of the
meristematic zone and also due to the reduction in
both new cell formation and cell elongation in the
extension region of the root.
Remarkable reductions of fresh and dry
weight were observed in shoot part in response to
arsenic. Similar results were observed by Jian et al.
(2008), when arsenic accumulates in root and of
rice grain which results in reduced yield;
Germination of two rice (Oryza sativa L.) cultivars
(Ratna and LR 36) in the presence of 10 mu M
PbCl2 and 10 mu M HgCl2 decreased germination
percentage, germination index, shoot/root length,
tolerance index and dry mass of shoots and roots
(Mishra and Choudhari, 1998) The effects were
more pronounced in tolerant cultivar IR 36 than in
the relatively susceptible cultivar Ratna.
The arsenic contamination lead to the
change of morphological characters of Salsola kali
was reported by Guadalupe et al. (1996);
Accumulation and distribution of arsenic
interrupted the physiological activities of tea plant
(Yuan, 2008); Azizur et al. (2007) reported that
arsenic toxicity affects the photosynthesis which
ultimately results in the reduction of rice growth
and yield ; Arsenic accumulation in rice inhibited
the metabolism of rice for which he suggested to
develop crop varieties to overcome the arsenic
contamination (Rakesh et al., 2010); Two varieties
of Cicer arietinum L. were tolerant to arsenic
accumulation in the root and shoot and detoxified
arsenic through chelation with GSH (Gupta et al.,
2008).
In an attempt to understand the molecular
basis of stress proteins, produced by the plants
under stress was detected using SDS-PAGE. The
protein was isolated from 30
th
day old plants.
Comparing the profiles of control and metal treated
rice plants with different concentrations of sodium
arsenate 25, 50 and 100 ppm, induced significant
changes in the patterns of proteins. These results
revealed that the proteins were expressed in specific
regions of rice plants adapted to metal stress (Fig.
1).
Out of 10 rice varieties, a total of 76, 68 and
50 protein bands were observed for 25, 50 and 100
ppm respectively, whereas control produced 86
protein bands with molecular weight ranging from
36.30 to 218.7 KD. In 25 ppm, proteins with
different molecular weight (165.95-27.54 KD) were
observed in each variety. In 50 ppm, protein bands
decreased when compared with control and 25 ppm,
and their molecular weight ranged from 218.77 to
Journal of Research in Agriculture (2011) 1: 011-016 013
Britto et al.,2011


23.98 KD. In 100 ppm, protein bands highly
decreased when compared with all other
concentration and control, and their molecular
weight ranged from 218.77 to 18.11 KD.
Proteins which are up regulated by stress
conditions (stress proteins) have been observed in
response to high and low temperatures, salinity,
droughts and several other stress factors (Pareek et
al., 1997). Large amount of protein observed in low
concentration may be ascribed to the inhibition of
protein synthesis under increasing concentration of
stress treatment. The number of polypeptides is
reduced in each concentration when compared to
control. Similar work was done in arsenic stress-
induced rice leaves in which differentially
expressed proteins were observed by Nagib Ahsan
et al. (2010) A total of 14 protein spots showed
reproducible changes in expression of at least 1.5-
fold when compared to the control and showed a
similar expression pattern in 50m and 100 M
treatments. In the present study, 68 protein bands in
50 ppm concentration and 50 bands in 100 ppm
concentrations were observed.
The rate of protein biosynthesis shows a
general decline during stress conditions. Despite
overall reduction in protein synthesis activity, it is
interesting to note that cell preferentially synthesize
stress protein. In certain cases stress proteins play a
crucial role in assisting the cell to carry out their
metabolic activities during adverse conditions
(Grover et al., 1993; Viswanathan and Khanna,
1996). The synthesis and accumulation of most of
the polypeptides in arsenic stress, in the present
study suggests major mechanisms that underlie
adaptation or tolerance to arsenic stress. It is
generally assumed that stress induced proteins play
a role in tolerance, but direct evidence is still
lacking and the function of many stress responsive
genes are unknown (Ingram and Bartels, 1996).
Earlier, Mani et al. (2010) identified a
dehydration responsive nuclear protein in rice and
Huang et al. (2009) cloned a Zinc finger protein
gene from rice and designated as SRZ1 (Stress
repressive Zinc finger protein 1). Stress associated
proteins are either synthesized de novo in response
to stress or present constitutively at low level and
their expression increases in response to stress (Arti
and Aruna, 2004)

CONCLUSION
From the present study it may be concluded
that rice plant induced with different concentrations
of arsenic reduced the growth leading to the
reduced productivity. But still IR45 variety was
found to be tolerant to the arsenic stress when
compared to the other varieties chosen. Thus this
variety seemed to have high tolerance towards
arsenic toxicity and may be grown in arsenic
contaminated areas without any major risk of
significant accumulation of arsenic in aerial parts.

ACKNOWLEDGEMENT
The authors are grateful to the Indian
Council of Medical Research, Government of India,
New Delhi (Ref: 59/12/2006/BMS/TRM dt.
26.03.2009) for financial support.

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016 Journal of Research in Agriculture (2011) 1: 011-016
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Sample
Conc. of
Arsenic
(ppm)
Shoot length (cm) Leaf area (cm
2
)
Root
length
(cm)
Fresh
wt (g)
Dry
wt (g)
15
th

day
20
th

Day
25
th

Day
30
th

day
15
th

day
20
th

day
25
th

day
30
th

day
IR 32 Control 9.2 11 12.3 14 1.2 1.76 2.2 2.4 5.5 1.410 0.638
25 8.5 9.0 9.5 10 1.72 1.68 1.35 0.92 5.5 1.512 0.412
50 8.5 8.8 9 9 1.35 0.97 0.83 0.47 5.1 1.420 0.828
100 9.4 9.5 9.5 9.5 1.12 1.01 0.96 0.48 4.8 1.528 0.292
IR 36 Control 10.3 12 13.2 15 1.17 1.68 1.80 2.45 4.5 1.431 0.670
25 9.5 10.2 10.5 11 1.64 1.72 1.38 0.92 5.3 1.448 0.412
50 9.5 9.7 9.9 10 1.42 1.23 0.55 0.43 4.9 1.397 0.350
100 11.4 11.5 11.6 11.6 1.14 0.78 0.42 0.39 4.7 1.448 0.312
IR 45 Control 13.3 14.5 11.1 18 1.84 1.84 2.5 2.75 4.4 1.528 0.618
25 12.5 14.1 14.5 15 1.38 1.47 1.13 1.06 5.1 1.510 0.432
50 12.6 12.8 13 13 1.47 1.41 1.32 0.98 4.9 1.423 0.398
100 12.4 12.5 12.5 12.6 2.15 1.72 1.32 0.88 4.6 1.391 0.360
IR 46 Control 12.8 14.8 15.4 17 1.84 1.84 2.08 2.35 4.3 1.632 0.712
25 12.5 13.5 13.8 14 1.88 1.88 1.5 1.41 4.9 1.574 0.412
50 13.8 14.9 14.9 15 1.26 1.26 0.88 0.43 4.6 1.549 0.398
100 13.0 13.5 13.5 13.5 1.12 0.90 0.68 0.41 4.7 1.519 0.352
IR 50 Control 10.5 11.2 12.1 13 1.20 1.60 2.25 2.60 4.7 1.502 0.643
25 9.8 10.2 10.5 10.8 1.68 1.54 1.38 0.93 5.3 1.439 0.433
50 10.2 10.5 10.5 10.5 1.54 1.23 0.86 0.48 5.1 1.397 0.339
100 9.0 9.2 9.2 9.2 1.17 1.02 0.78 0.46 4.8 1.358 0.298
ADT32 Control 12.8 13.8 14.3 16 1.30 1.60 1.76 2.30 4.8 1.328 0.518
25 12.4 13.2 13.5 14.5 1.83 1.62 1.47 1.38 5.5 1.382 0.419
50 13.0 13.5 14 14 1.74 1.57 1.44 0.80 5.2 1.405 0.314
100 10.7 10.8 11 11 1.60 1.38 0.92 0.48 4.6 1.492 0.293
ADT 39 Control 10.0 11.1 12 13 1.17 1.56 1.76 1.88 4.7 1.650 0.712
25 9.5 10.0 10.5 11 1.56 1.43 1.38 1.35 5.2 1.682 0.687
50 9.2 9.6 9.9 9.8 1.48 1.14 0.98 0.52 5.1 1.593 0.428
100 8.7 8.9 9 9 1.32 1.09 0.90 0.46 4.4 1.584 0.390
CO45 Control 10.4 11.3 12.1 13 1.64 1.83 2.25 2.60 5.0 1.238 0.512
25 7.5 8.2 8.5 8.5 1.68 1.35 1.26 1.00 5.5 1.259 0.403
50 9.0 9.5 10 10 1.59 1.26 0.94 0.53 5.2 1.205 0.321
100 9.4 9.5 9.5 9.5 1.35 1.17 0.82 0.47 5.1 1.329 0.282
Ambai 16 Control 12.0 13.5 14.5 16 1.29 1.72 2.28 2.52 4.7 1.325 0.502
25 11.3 12.0 12.2 13 1.32 1.21 1.13 0.98 5.1 1.438 0.418
50 10.2 10.4 11.5 11.5 1.17 0.98 0.94 0.57 5.0 1.392 0.321
100 11.4 11.5 11.5 11.6 1.08 0.92 0.72 0.43 4.9 1.486 0.243
Singapore Control 11.6 12.8 13.7 15 1.64 1.92 2.05 2.5 4.8 1.486 0.670
25 11.0 12.0 12.6 13 1.35 1.29 1.11 1.0 4.8 1.592 0.412
50 10.1 10.3 10.3 10.3 1.21 0.96 0.90 0.49 4.9 1.436 0.350
100 9.4 9.5 9.5 9.5 1.17 0.93 0.86 0.43 5.0 1.588 0.292
Table 1. Effect of arsenic stress on shoot length, Leaf area, Root length, Fresh and Dry weight of the ten rice varieties